Sander S. Rensen

Regulation and characteristics of vascular smooth muscle cell phenotypic diversity

Vascular smooth muscle cells can perform both contractile and synthetic functions, which are associated with and characterised by changes in morphology, proliferation and migration rates, and the expression of different marker proteins. The resulting phenotypic diversity of smooth muscle cells appears to be a function of innate genetic programmes and environmental cues, which include biochemical factors, extracellular matrix components, and physical factors such as stretch and shear stress. Because of the diversity among smooth muscle cells, blood vessels attain the flexibility that is necessary to perform efficiently under different physiological and pathological conditions. In this review, we discuss recent literature demonstrating the extent and nature of smooth muscle cell diversity in the vascular wall and address the factors that affect smooth muscle cell phenotype. (Neth Heart J 2007;15:100-8.)

Trans-eQTLs Reveal That Independent Genetic Variants Associated with a Complex Phenotype Converge on Intermediate Genes, with a Major Role for the HLA

For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10−16). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes.

Unraveling the regulatory mechanisms underlying tissue-dependent genetic variation of gene expression.

It is known that genetic variants can affect gene expression, but it is not yet completely clear through what mechanisms genetic variation mediate this expression. We therefore compared the cis-effect of single nucleotide polymorphisms (SNPs) on gene expression between blood samples from 1,240 human subjects and four primary non-blood tissues (liver, subcutaneous, and visceral adipose tissue and skeletal muscle) from 85 subjects. We characterized four different mechanisms for 2,072 probes that show tissue-dependent genetic regulation between blood and non-blood tissues: on average 33.2% only showed cis-regulation in non-blood tissues; 14.5% of the eQTL probes were regulated by different, independent SNPs depending on the tissue of investigation. 47.9% showed a different effect size although they were regulated by the same SNPs. Surprisingly, we observed that 4.4% were regulated by the same SNP but with opposite allelic direction. We show here that SNPs that are located in transcriptional regulatory elements are enriched for tissue-dependent regulation, including SNPs at 3′ and 5′ untranslated regions (P = 1.84×10−5 and 4.7×10−4, respectively) and SNPs that are synonymous-coding (P = 9.9×10−4). SNPs that are associated with complex traits more often exert a tissue-dependent effect on gene expression (P = 2.6×10−10). Our study yields new insights into the genetic basis of tissue-dependent expression and suggests that complex trait associated genetic variants have even more complex regulatory effects than previously anticipated.

Human intestinal microbiota composition is associated with local and systemic inflammation in obesity

Intestinal microbiota have been suggested to contribute to the development of obesity, but the mechanism remains elusive. The relationship between microbiota composition, intestinal permeability, and inflammation in nonobese and obese subjects was investigated.

Neutrophil Activation in Morbid Obesity, Chronic Activation of Acute Inflammation

Recent studies show that morbid obesity is associated with activation of the innate immune response. Neutrophil activation is a fundamental process in the innate immune response. Therefore, the activation state of neutrophils in severely obese subjects and the effect of bariatric surgery on neutrophil activation was evaluated. Neutrophil activation was assessed by measuring circulating concentrations of myeloperoxidase (MPO) and calprotectin in 37 severely obese and 9 control subjects (enzyme‐linked immunosorbent assay). Moreover, membrane expression of CD66b on circulating neutrophils was measured using flow cytometry in a group of seven severely obese and six control subjects. Immunohistochemical detection of MPO was performed in adipose and muscle tissue. Plasma MPO and calprotectin levels were significantly increased in severely obese subjects as compared to healthy controls, 27.1 ± 10.8 vs. 17.3 ± 5.5 ng/ml (P < 0.001) and 115.5 ± 43.5 vs. 65.1 ± 23.1 ng/ml (P < 0.001) for MPO and calprotectin, respectively. In line, CD66b expression was significantly increased in severely obese individuals, 177.3 ± 43.7 vs. 129.7 ± 9.2 (mean fluorescence intensity) (P < 0.01). Bariatric surgery resulted in decreased calprotectin, but MPO plasma levels remained elevated. Adipose and muscle tissue did not contain increased numbers of MPO expressing cells in severely obese individuals. These results point out that circulating neutrophils are activated to a greater extent in severely obese subjects. Our data support the finding that the innate immune system is activated in severely obese individuals. Moreover, because neutrophils have a short life span, this indicates that the chronic inflammatory condition associated with morbid obesity is characterized by a continuous activation of the innate immune system.

Cultured Porcine Coronary Artery Smooth Muscle Cells: A New Model With Advanced Differentiation

Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage after enzymatic digestion of the media. The effects of heparin, transforming growth factor (TGF)-beta1 or TGF-beta2, and all-trans-retinoic acid (tRA) on proliferation, migration, and differentiation of these cells also were examined. Porcine arterial SMCs in culture not only express high levels of alpha-smooth muscle (SM) actin but, contrary to rodent SMCs, also maintain an appreciable expression of SM myosin heavy chain isoforms 1 and 2, desmin, and smoothelin, a recently described late differentiation marker of vascular SMCs. We demonstrate for the first time that smoothelin is colocalized with alpha-SM actin in these cells. Finally, we show that in the porcine model, heparin is more potent than TGF-beta1 or TGF-beta2 and tRA in terms of inhibition of proliferation and migration and of increasing the expression of differentiation markers. This model should be a useful complement to in vivo studies of SMC differentiation and of pathological situations such as restenosis and atheromatosis.

De novo lipogenesis in human fat and liver is linked to ChREBP-β and metabolic health

Clinical interest in de novo lipogenesis has been sparked by recent studies in rodents demonstrating that de novo lipogenesis specifically in white adipose tissue produces the insulin-sensitizing fatty acid palmitoleate. By contrast, hepatic lipogenesis is thought to contribute to metabolic disease. How de novo lipogenesis in white adipose tissue versus liver is altered in human obesity and insulin resistance is poorly understood. Here we show that lipogenic enzymes and the glucose transporter-4 are markedly decreased in white adipose tissue of insulin-resistant obese individuals compared with non-obese controls. By contrast, lipogenic enzymes are substantially upregulated in the liver of obese subjects. Bariatric weight loss restored de novo lipogenesis and glucose transporter-4 gene expression in white adipose tissue. Notably, lipogenic gene expression in both white adipose tissue and liver was strongly linked to the expression of carbohydrate-responsive element-binding protein-β and to metabolic risk markers. Thus, de novo lipogenesis predicts metabolic health in humans in a tissue-specific manner and is likely regulated by glucose-dependent carbohydrate-responsive element-binding protein activation.

Increased Hepatic Myeloperoxidase Activity in Obese Subjects with Nonalcoholic Steatohepatitis

Inflammation and oxidative stress are considered critical factors in the progression of nonalcoholic fatty liver disease. Myeloperoxidase (MPO) is an important neutrophil enzyme that can generate aggressive oxidants; therefore, we studied the association between MPO and nonalcoholic fatty liver disease. The distribution of inflammatory cells containing MPO in liver biopsies of 40 severely obese subjects with either nonalcoholic steatohepatitis (NASH) (n = 22) or simple steatosis (n = 18) was investigated by immunohistochemistry. MPO-derived oxidative protein modifications were identified by immunohistochemistry and correlated to hepatic gene expression of CXC chemokines and M1/M2 macrophage markers as determined by quantitative PCR. MPO plasma levels were determined by ELISA. The number of hepatic neutrophils and MPO-positive Kupffer cells was increased in NASH and was accompanied by accumulation of hypochlorite-modified and nitrated proteins, which can be generated by the MPO-H2O2 system. Liver CXC chemokine expression was higher in patients with accumulation of MPO-mediated oxidation products and correlated with hepatic neutrophil sequestration. Plasma MPO levels were elevated in NASH patients. Interestingly, neutrophils frequently surrounded steatotic hepatocytes, resembling the crown-like structures found in obese adipose tissue. Furthermore, hepatic M2 macrophage marker gene expression was increased in NASH. Our data indicate that accumulation of MPO-mediated oxidation products, partly derived from Kupffer cell MPO, is associated with induction of CXC chemokines and hepatic neutrophil infiltration and may contribute to the development of NASH.

The Role of Microbial Amino Acid Metabolism in Host Metabolism

Disruptions in gut microbiota composition and function are increasingly implicated in the pathogenesis of obesity, insulin resistance, and type 2 diabetes mellitus. The functional output of the gut microbiota, including short-chain fatty acids and amino acids, are thought to be important modulators underlying the development of these disorders. Gut bacteria can alter the bioavailability of amino acids by utilization of several amino acids originating from both alimentary and endogenous proteins. In turn, gut bacteria also provide amino acids to the host. This could have significant implications in the context of insulin resistance and type 2 diabetes mellitus, conditions associated with elevated systemic concentrations of certain amino acids, in particular the aromatic and branched-chain amino acids. Moreover, several amino acids released by gut bacteria can serve as precursors for the synthesis of short-chain fatty acids, which also play a role in the development of obesity. In this review, we aim to compile the available evidence on the contribution of microbial amino acids to host amino acid homeostasis, and to assess the role of the gut microbiota as a determinant of amino acid and short-chain fatty acid perturbations in human obesity and type 2 diabetes mellitus.

Activation of the complement system in human nonalcoholic fatty liver disease

Activation of the innate immune system plays a major role in nonalcoholic fatty liver disease (NAFLD). The complement system is an important component of innate immunity that recognizes danger signals such as tissue injury. We aimed to determine whether activation of the complement system occurs in NAFLD, to identify initiating pathways, and to assess the relation between complement activation, NAFLD severity, apoptosis, and inflammatory parameters. Liver biopsies of 43 obese subjects with various degrees of NAFLD and of 10 healthy controls were analyzed for deposition of complement factors C1q, mannose‐binding lectin (MBL), C4d, activated C3, and membrane attack complex (MAC)‐associated C9. Furthermore, hepatic neutrophil infiltration, apoptosis, and pro‐inflammatory cytokine expression were quantified. Whereas complement activation was undetectable in the liver of healthy subjects, 74% of the NAFLD patients showed hepatic deposition of activated C3 and C4d. C1q as well as MBL accumulation was found in most activated C3‐positive patients. Strikingly, 50% of activated C3‐positive patients also displayed MAC‐associated C9 deposition. Deposition of complement factors was predominantly seen around hepatocytes with macrovesicular steatosis. Subjects showing accumulation of activated C3 displayed increased numbers of apoptotic cells. Importantly, hepatic neutrophil infiltration as well as interleukin (IL)‐8 and IL‐6 expression was significantly higher in patients showing activated C3 deposition, whereas patients with C9 deposition additionally had increased IL‐1β expression. Moreover, nonalcoholic steatohepatitis (NASH) was more prevalent in patients showing hepatic C9 or activated C3 deposition. Conclusion: There is widespread activation of the complement system in NAFLD, which is associated with disease severity. This may have important implications for the pathogenesis and progression of NAFLD given the function of complement factors in clearance of apoptotic cells, hepatic fibrosis, and liver regeneration. (HEPATOLOGY 2009.)